The transcriptome of primary colon cancer: In order to characterize patterns of global transcriptional deregulation in primary colon carcinomas, we performed gene expression profiling of 73 tumors (UICC stage II, n=33 and UICC stage III, n=40) using oligonucleotide microarrays. For 30 of the tumors, expression profiles were compared to those from matched normal mucosa samples. We identified a set of 1,950 genes with highly significant deregulation between tumors and mucosa samples (P&lt;1e-7). A significant proportion of these genes mapped to chromosome 20 (P=0.01). Seventeen genes had a greater than five-fold average expression difference between normal colon mucosa and carcinomas, including up-regulation of MYC and of HMGA1, a putative oncogene. Furthermore, we identified 68 genes that were significantly differentially expressed between lymph node negative and positive tumors (P&lt;0.001), the functional annotation of which revealed a preponderance of genes that play a role in cellular immune response and surveillance. The microarray-derived gene expression levels of 20 deregulated genes were validated using quantitative real-time RT-PCR in more than 40 tumor and normal mucosa samples with good concordance between the techniques. Finally, we established a relationship between specific genomic imbalances, which were mapped for 32 of the analyzed colon tumors by comparative genomic hybridization, and alterations of global transcriptional activity. Previously, we had conducted a similar analysis of primary rectal carcinomas. The systematic comparison of colon and rectal carcinomas revealed a significant overlap of genomic imbalances and transcriptional deregulation, including activation of the Wnt/b-catenin signaling cascade, suggesting similar pathogenic pathways. The transcriptome of primary rectal adenocarcinomas: In order to identify genetic alterations underlying rectal carcinogenesis, we used global gene expression profiling of a series of 17 locally advanced rectal adenocarcinomas and 20 normal rectal mucosa biopsies on oligonucleotide arrays. A total of 351 genes were differentially expressed (p&lt; 1.0e-7) between normal rectal mucosa and rectal carcinomas, 77 genes had a greater than five-fold difference, and 85 genes always had at least a two-fold change in all of the matched samples. 12 genes satisfied all three of these criteria. Altered expression of genes such as PTGS2 (COX2), WNT1, TGFB1, VEGF and MYC was confirmed, while our data for other genes like PPARD and LEF1 were inconsistent with previous reports. In addition, we found deregulated expression of many genes whose involvement in rectal carcinogenesis has not been reported. By mapping the genomic imbalances in the tumors using comparative genomic hybridization, we could show that DNA copy number gains of recurrently aneuploid chromosome arms 7p, 8q, 13q, 18q, 20p and 20q correlated significantly with their average chromosome arm expression profile. Taken together, our results demonstrate that both the high-level, significant transcriptional deregulation of specific genes and general modification of the average transcriptional activity of genes residing on aneuploid chromosomes coexist in rectal adenocarcinomas. Identification of a putative oncogene in 13q involved in colorectal cancer: Chromosome 13 is one of the most commonly gained chromosomes in colorectal carcinomas. Despite the prevalence of this aneuploidy, it is not clear which gene or genes are the targets of this chromosomal imbalance. We have therefore analyzed a set of 73 colon cancer samples using a NCI oligonucleotide arrays (22K) using the Operon V2 oligo set (see above). A previous study using rectal cancer showed that a high number of differentially expressed (upregulated) genes mapped to chromosome 13. We also performed array CGH in a set of 34 tumors included in the previous study. For this purpose, we used a high-resolution oligonucleotide-based microarray (185K) from Agilent. From this study, we were able to separate a set of tumors that show localized amplifications on specific chromosomal bands. This allowed identification of regions of minimal overlap on this chromosome. The concordant analysis of a significant number of samples using aCGH and expression profiling reduced the number of potential candidate genes to a list of some 39 genes residing on chromosome 13, whose functional analysis in colorectal cancers is now underway. These efforts resulted in the identification of some 11 genes whose down-regulation profoundly reduced the viability of colorectal cancer cell lines. We are now in the process of conducting time course experiments after knockdown to map the functional space in which these genes operate, and to identify small molecules that show similar gene expression changes as the siRNA mediated knockdown. Apoptosis screen in colorectal cancer cell lines: One of the pathways of malignant transformation includes the ability of cancerous cells to avoid apoptosis. We are currently exploring the role of genes that regulate apoptosis in colorectal cancer cells. Using a commercial set of siRNAs, we have conducted screens of siRNAs targeting 418 genes considered to have a role in apoptosis. Two siRNAs were examined for each gene and the screen was performed using the colorectal cancer cell line SW480. The effect of each siRNA was evaluated with a viability assay. In order to identify potentially novel as well as combination therapeutic interventions, we used RNAi with and without the presence of 5-FU, a chemotherapeutic agent used for the treatment of colon cancer. Cells were seeded on a complex of siRNA and cationic lipid in 96-well plates for reverse transfection. When appropriate, 5-FU (80M corresponding to the EC50) was added after 48 hours, and cell viability was assayed after a further 72 hours of incubation. The addition of 5-FU uniformly decreased the viability of the cells by approximately 50%. We identified numerous genes that further increased the sensitivity of SW480 cells to 5-FU. Stringent filtering of decreased viability by siRNAs in the absence of 5-FU treatment led us to focus on two genes as potential novel targets that reduce cell viability: a gene that belongs to the class of nucleoporins, and a caspase-associated gene. The siRNAs to both genes reduced SW480 viability by greater than 40%. We have fully characterized the reduction in mRNA levels mediated by the siRNAs corresponding to these two genes and consistently observe that their down-regulation reduces the viability of SW480 cells. As a further validation of their effect, we have silenced the caspase-associated gene in additional cell lines. The reduction in cell viability was more pronounced in another aneuploid cell line, SW837, than in a diploid cell line, SW48, indicating a potential susceptibility of aneuploid cell lines to the down-regulation of this target gene. We are currently investigating the functional mechanism responsible for this effect.